Tottering (Tg) mice carry the mutation P601L in their Cacna1a encoded Cav2.1 channels. Transmitter release at the wild-type neuromuscular junction (NMJ) is almost exclusively mediated by Cav2.1 channels, and we used this model synapse to study synaptic consequences of the Tg mutation. With electrophysiology, and using subtype-specific Cav2 channel-blocking toxins, we assessed a possible compensatory contribution of non-Cav2.1 channels to evoked acetylcholine (ACh) release at Tg NMJs. Release was reduced by approximately 75% by the Cav2.1 channel blocker omega-agatoxin-IVA, which was less than the approximately 95% reduction observed in wild-type. Release at Tg NMJs, but not at wild-type synapses, was reduced by approximately 15% by SNX-482, a Cav2.3 channel blocker. No Cav2.2 channel involvement was found. Probably, there is a small reduction in functional presynaptic Cav2.1 channels at Tg NMJs, which is compensated for by Cav2.3 channels. The remaining Cav2.1 channels are likely to convey enlarged Ca2+ flux, because evoked ACh release at Tg NMJs, at low extracellular Ca2+ concentration, was approximately sixfold higher than at wild-type NMJs. This is the first report of compensatory expression of non-Cav2.1 channels at NMJs of mice with a single amino acid change in Cav2.1.